Name: studying rna interactors of protein kinase rna-activated during the mammalian cell cycle
Uploaded: 2019-03-05T14:00:00
Description: Watch this Scientific Journal Video about Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle at JoVE.com

这项工作得到了韩国国家研究基金会通过韩国政府科学和信息通信技术部资助的韩国国家研究基金会 (nrf-2016r1c1b29886) 的支持。

1. 溶液和细胞制备
<ol><li>解决方案准备<ol>
<li>对于细胞培养培养基, 在杜尔贝科改良鹰培养基 (dmem) 的500毫升中加入50毫升的胎儿牛血清 (fbs), 制备平细胞培养培养基。 注: 抗生素可以添加到细胞培养基中, 但我们不使用抗生素。</li>
		<li>对于0.1% 的甲醛, 在1x 磷酸盐缓冲盐水 (pbs) 中溶解 4% (w/v), 在热板上加热并稀释, 通过添加 1x pbs 使30% 毫升的甲醛。 注意: 在烟罩中执行所有步骤, 并注意不要?...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interferon-inducible+double-stranded+RNA-activated+protein+kinase+self-associates+in+vitro+and+in+vivo.">The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo.</a> Proceedings of the National Academy of Sciences. 92 (18), 8283-8287 (1995).</li><li>Bennett, R. L., Pan, Y., Christian, J., Hui, T., May, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RAX/PACT-PKR+stress+response+pathway+promotes+p53+sumoylation+and+activation,+leading+to+G(1)+arrest.">The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G(1) arrest.</a> Cell Cycle. 11 (1), 407-417 (2012).</li><li>Yang, X., Nath, A., Opperman, M. J., Chan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+double-stranded+RNA-dependent+protein+kinase+differentially+regulates+insulin+receptor+substrates+1+and+2+in+HepG2+cells.">The double-stranded RNA-dependent protein kinase differentially regulates insulin receptor substrates 1 and 2 in HepG2 cells.</a> Molecular and Cellular Biology. 21 (19), 3449-3458 (2010).</li><li>Zamanian-Daryoush, M., Mogensen, T. H., DiDonato, J. A., Williams, B. R. 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Oncogene. 26 (8), 1201-1212 (2007).</li><li>Dabo, S., Meurs, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dsRNA-dependent+protein+kinase+PKR+and+its+role+in+stress,+signaling+and+HCV+infection.">dsRNA-dependent protein kinase PKR and its role in stress, signaling and HCV infection.</a> Viruses. 4 (11), 2598-2635 (2012).</li><li>Black, T. L., Safer, B., Hovanessian, A., Katze, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cellular+68,000-Mr+Protein-Kinase+Is+Highly+Autophosphorylated+and+Activated+yet+Significantly+Degraded+during+Poliovirus+Infection+-+Implications+for+Translational+Regulation.">The Cellular 68,000-Mr Protein-Kinase Is Highly Autophosphorylated and Activated yet Significantly Degraded during Poliovirus Infection - Implications for Translational Regulation.</a> Journal of Virology. 63 (5), 2244-2251 (1989).</li><li>Bando, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+RNA+dependent+protein+kinase+(PKR)+is+involved+in+the+extrastriatal+degeneration+in+Parkinson's+disease+and+Huntington's+disease.">Double-strand RNA dependent protein kinase (PKR) is involved in the extrastriatal degeneration in Parkinson's disease and Huntington's disease.</a> Neurochemistry International. 46 (1), 11-18 (2005).</li><li>Onuki, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA-dependent+protein+kinase+is+involved+in+tunicamycin-induced+apoptosis+and+Alzheimer's+disease.">An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease.</a> The EMBO Journal. 23 (4), 959-968 (2004).</li><li>Peel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+cell+stress+kinase+PKR+in+Alzheimer's+disease+and+human+amyloid+precursor+protein+transgenic+mice.">Activation of the cell stress kinase PKR in Alzheimer's disease and human amyloid precursor protein transgenic mice.</a> Neurobiology of Disease. 14 (1), 52-62 (2003).</li><li>Peel, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA-dependent+protein+kinase,+PKR,+binds+preferentially+to+Huntington's+disease+(HD)+transcripts+and+is+activated+in+HD+tissue.">Double-stranded RNA-dependent protein kinase, PKR, binds preferentially to Huntington's disease (HD) transcripts and is activated in HD tissue.</a> Human Molecular Genetics. 10 (15), 1531-1538 (2001).</li><li>Suen, K. C., Yu, M. S., So, K. F., Chang, R. C., Hugon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upstream+signaling+pathways+leading+to+the+activation+of+double-stranded+RNA-dependent+serine/threonine+protein+kinase+in+beta-amyloid+peptide+neurotoxicity.">Upstream signaling pathways leading to the activation of double-stranded RNA-dependent serine/threonine protein kinase in beta-amyloid peptide neurotoxicity.</a> Journal of biological chemistry. 278 (50), 49819-49827 (2003).</li><li>Nakamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+PKR+complexes+with+TRBP+in+Immunometabolic+regulation+and+eIF2alpha+phosphorylation+in+obesity.">A critical role for PKR complexes with TRBP in Immunometabolic regulation and eIF2alpha phosphorylation in obesity.</a> Cell Reports. 11 (2), 295-307 (2015).</li><li>Saito, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+interferon-induced+double-stranded+RNA-dependent+protein+kinase+activity+by+Sindbis+virus+infection+and+heat-shock+stress.">Enhancement of the interferon-induced double-stranded RNA-dependent protein kinase activity by Sindbis virus infection and heat-shock stress.</a> Microbiology and Immunology. 34 (10), 859-870 (1990).</li><li>Youssef, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+for+snoRNAs+in+PKR+activation+during+metabolic+stress.">Potential role for snoRNAs in PKR activation during metabolic stress.</a> Proceedings of the National Academy of Sciences. 112 (16), 5023-5028 (2015).</li><li>Murtha-Riel, P., Davies, M. V., Choi, S. Y., Hershey, J. W., Kaufman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+Phosphorylation-resistant+Eukaryotic+Initiation+Factor+2+a-Subunit+Mitigates+Heat+Shock+Inhibition+of+Protein+Synthesis.">Expression of a Phosphorylation-resistant Eukaryotic Initiation Factor 2 a-Subunit Mitigates Heat Shock Inhibition of Protein Synthesis.</a> The Journal of Biological Chemistry. 268, 12946-12951 (1993).</li><li>Benkirane, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+potential+of+TAR+RNA+binding+protein+TRBP+and+its+regulatory+interaction+with+RNA-dependent+protein+kinase+PKR.">Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR.</a> The EMBO Journal. 16 (3), 611-624 (1997).</li><li>Koromilas, A., Roy, S., Barber, G., Katze, M., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+transformation+by+a+mutant+of+the+IFN-inducible+dsRNA-dependent+protein+kinase.">Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase.</a> Science. 257 (5077), 1685-1689 (1992).</li><li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKR+is+activated+by+cellular+dsRNAs+during+mitosis+and+acts+as+a+mitotic+regulator.">PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator.</a> Genes &amp; Development. 28 (12), 1310-1322 (2014).</li><li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+human+tarbp2+reveals+cellular+microRNA+targets+and+cell-cycle+function+of+TRBP.">Deletion of human tarbp2 reveals cellular microRNA targets and cell-cycle function of TRBP.</a> Cell Reports. 9 (3), 1061-1074 (2014).</li><li>Zhu, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+PKR+promotes+network+excitability+and+enhanced+cognition+by+interferon-gamma-mediated+disinhibition.">Suppression of PKR promotes network excitability and enhanced cognition by interferon-gamma-mediated disinhibition.</a> Cell. 147 (6), 1384-1396 (2011).</li><li>Dagon, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA-dependent+protein+kinase,+PKR,+down-regulates+CDC2/cyclin+B1+and+induces+apoptosis+in+non-transformed+but+not+in+v-mos+transformed+cells.">Double-stranded RNA-dependent protein kinase, PKR, down-regulates CDC2/cyclin B1 and induces apoptosis in non-transformed but not in v-mos transformed cells.</a> Oncogene. 20 (56), 8045-8056 (2001).</li><li>Elbarbary, R. A., Li, W., Tian, B., Maquat, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAU1+binding+3'+UTR+IRAlus+complements+nuclear+retention+to+protect+cells+from+PKR-mediated+translational+shutdown.">STAU1 binding 3' UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown.</a> Genes &amp; Development. 27 (13), 1495-1510 (2013).</li><li>Cho, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIN28A+is+a+suppressor+of+ER-associated+translation+in+embryonic+stem+cells.">LIN28A is a suppressor of ER-associated translation in embryonic stem cells.</a> Cell. 151 (4), 765-777 (2012).</li><li>Licatalosi, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HITS-CLIP+yields+genome-wide+insights+into+brain+alternative+RNA+processing.">HITS-CLIP yields genome-wide insights into brain alternative RNA processing.</a> Nature. 456 (7221), 464-469 (2008).</li><li>Van Nostrand, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+transcriptome-wide+discovery+of+RNA-binding+protein+binding+sites+with+enhanced+CLIP+(eCLIP).">Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).</a> Nature Methods. 13 (6), 508-514 (2016).</li><li>Kim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKR+Senses+Nuclear+and+Mitochondrial+Signals+by+Interacting+with+Endogenous+Double-Stranded+RNAs.">PKR Senses Nuclear and Mitochondrial Signals by Interacting with Endogenous Double-Stranded RNAs.</a> Molecular Cell. 71 (6), 1051-1063 (2018).</li><li>Kim, B., Jeong, K., Kim, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+Mapping+of+DROSHA+Cleavage+Sites+on+Primary+MicroRNAs+and+Noncanonical+Substrates.">Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates.</a> Molecular Cell. 66 (2), 258-269 (2017).</li><li>Ricci, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staufen1+senses+overall+transcript+secondary+structure+to+regulate+translation.">Staufen1 senses overall transcript secondary structure to regulate translation.</a> Nature Structural &amp; Molecular Biology. 21 (1), 26-35 (2014).</li><li>Kim, B., Kim, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fCLIP-seq+for+transcriptomic+footprinting+of+dsRNA-binding+proteins:+Lessons+from+DROSHA.">fCLIP-seq for transcriptomic footprinting of dsRNA-binding proteins: Lessons from DROSHA.</a> Methods. , (2018).</li><li>Dhir, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+double-stranded+RNA+triggers+antiviral+signalling+in+humans.">Mitochondrial double-stranded RNA triggers antiviral signalling in humans.</a> Nature. 560 (7717), 238-242 (2018).</li></ol>

图 1显示了制备 s 或 m 相抑制样本的过程。为了在 s 期的细胞中, 我们使用了胸苷双块法, 我们用胸苷对细胞进行了两次处理, 中间释放了 9小时, 以确保较高的逮捕效率 (图 1 a)。对于 m 期停止, 我们用胸苷治疗细胞一次, 然后释放 9小时, 然后应用诺康唑在启动基中阻断细胞 (图 1b)。准备细胞周期样本的一个关键步骤是在第一个胸苷?...

蛋白激酶 rna 激活 (pkr), 也被称为真核起始因子2α激酶 2 (EIF2AK2), 是一种特征良好的蛋白激酶, 传输 rna 提供的信息。它属于真核翻译起始2亚基α (eif2α) 激酶家族和磷酸酯 eif2α在丝氨酸51响应感染,以抑制全球翻译1。在此背景下, pkr 被病毒双链 rna (dsrna) 激活, 为 pkr 二聚和自磷酸化2提供了一个平台。除了 eif2α, pkr 还可以磷酸化 p53, 胰岛素受体底物 1, 抑制剂 b, 和 c-jun n-端子激酶 (jnk) 调节活性的许多信号转导途径 3,4,5, 6。
pkr 最初被确定为一种在脊髓灰质炎病毒感染过程中磷酸化 eif2α的激酶, 通过识别脊髓灰质炎病毒的 dsrna 7,8。pkr 越来越多地被发现在免疫反应之外发挥着多方面的作用, 其异常激活或故障在许多人类疾病中都隐含着。在细胞凋亡过程中经常观察到活着的磷酸化 pPKR (ppkr), 是退行性疾病患者的共同特征, 尤其是亨廷顿病、帕金森症和阿尔茨海默病等神经退行性疾病9 10,11,12, 13.此外, pkr 在代谢应激和热休克等各种压力条件下被激活, 14、15、16、17。另一方面, pkr 的抑制导致细胞增殖甚至恶性转化的增加 18,19。pPKR 功能在正常大脑功能和细胞周期中也很重要, 因为在 m 期20、21、22 期间, ppkr 水平升高。在这种情况下, ppkr 抑制全局转换, 并为正确的细胞分裂20所需的关键有丝分裂信号系统提供提示。此外, 长时间激活 pkr 导致 g2m 相细胞周期停止在中国仓鼠卵巢 23.因此, pkr 磷酸化由负反馈回路调节, 以确保在 mg1 过渡期间快速失活21。
尽管 pkr 功能范围广泛, 但由于缺乏能够激活 pkr 的标准化高吞吐量实验方法来捕获和识别 dsrna, 我们对 pkr 激活的理解有限。先前的研究表明, pkr 可以与由两个倒置 alu 重复 (iralus)20、24形成的 dsrna 相互作用, 但存在额外的细胞 dsrna 的可能性, 这些细胞 dsrna 可以在细胞周期内或以下激活 pkr。人类细胞的应激条件尚未探索。识别 rna 结合蛋白 (rbp) rna 相互体的常规方法是利用紫外光将 rna-rbp 复合物交联 25、26、27。最近的一项研究将这种紫外交联方法应用于小鼠系统, 并确定小核子蛋白 rna 可以调节 pkr 激活在代谢应激16。利用甲醛的高交联效率, 提出了一种在 hela 细胞28细胞周期中识别 pkr 相互作用 rna 的替代方法。在其他 dsRBPs (如 staufen 和 drosha29、30、31) 的研究中也采用了类似的方法。我们发现 pkr 可以与各种类型的非编码 rna 相互作用, 如短插层核元件 (sine)、长穿插核元素 (line)、内源性逆转录病毒元素 (erv), 甚至α-卫星 rna。此外, 我们还表明 pkr 可以与线粒体 rna (mtrna) 相互作用, 线粒体 rna 通过重链和光链 rna28之间的互补相互作用形成分子间 dsrna。最近的一份出版物进一步支持了我们的数据, 即一些 mtrna 以双工形式存在, 并可以激活 dsrna 传感器, 如黑色素瘤差异相关的蛋白质 5, 以诱导干扰素32。更重要的是, mtrna 的表达和亚细胞定位在细胞周期和各种压力源中被调节, 这可能对它们调节 pkr 激活28的能力很重要。
在本文中, 我们提出了一个详细的协议, 最近开发的甲醛交联和免疫沉淀 (fCLIP) 方法来捕获和分析 pkr 相互作用的 rna 在细胞周期。我们演示了用胸苷和诺加唑制备细胞周期抑制样本的方法。然后, 我们提出了分离 pkr 绑定 rna 的 fclip 过程, 以及一种准备高吞吐量测序库以识别这些 rna 的方法。此外, 我们还描述了使用 qrt-pcr 分析 pkr 绑定 rna 的详细过程。具体而言, 我们提出了一个字符串特定的逆转录过程来分析 mtrna 的字符串。所述协议针对 hara 细胞和 pkr 进行了优化, 但可轻松修改细胞周期样本、fclip 和特异性 qrt-pcr 分析等关键步骤, 以研究细胞 dsrna 或识别其他 dsRNAs 的 rna 干扰体。

<table>
	<tbody>
		<tr>
			<td>0.5 M EDTA, pH 8.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris, pH 7.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9855G</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris, pH 8.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9855G</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Nonidet-p40 (NP-40)</td>
			<td>Biosolution</td>
			<td>BN015</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Urea-acrylamide gel solution</td>
			<td></td>
			<td></td>
			<td>7 M (w/v) Urea and 0.5X TBE, stored protected from light at 4 &#176;C</td>
		</tr>
		<tr>
			<td>10X DNA loading buffer</td>
			<td>TaKaRa</td>
			<td>9157</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>3&#39; adaptor</td>
			<td></td>
			<td></td>
			<td>5&#39;-rApp NN NNT GGA ATT CTC GGG TGC CAA GG/3ddC/-3&#39;</td>
		</tr>
		<tr>
			<td>3 M Sodium Acetate pH 5.5</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9740</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#39; adaptor</td>
			<td></td>
			<td></td>
			<td>5&#39;-GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN-3&#39;</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid-phenol chloroform, pH 4.5</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9722</td>
			<td></td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Magnetic beads DNA/RNA clean up</td>
		</tr>
		<tr>
			<td>Antarctic alkaline phosphatase</td>
			<td>New England Biolabs</td>
			<td>M0289S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-DGCR8</td>
			<td></td>
			<td></td>
			<td>Made in house</td>
		</tr>
		<tr>
			<td>Anti-PKR (D7F7)</td>
			<td>Cell signaling technology</td>
			<td>12297S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PKR (Milli)</td>
			<td>Millipore EMD</td>
			<td>07-151</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP (100 mM)</td>
			<td>GE Healthcare</td>
			<td>GE27-2056-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue sodium salt</td>
			<td>Sigma-aldrich</td>
			<td>B5525</td>
			<td></td>
		</tr>
		<tr>
			<td>Calf intestinal alkaline phosphatase</td>
			<td>TaKaRa</td>
			<td>2250A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper 25 cm 2-position</td>
			<td>Sarstedt</td>
			<td>83.183</td>
			<td></td>
		</tr>
		<tr>
			<td>CMV promoter sequence</td>
			<td></td>
			<td></td>
			<td>5&#39;-CGCAAATGGGCGGTAGGCGTG-3&#39;</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium</td>
			<td>Welgene</td>
			<td>LM001-05</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mixture (2.5 mM)</td>
			<td>TaKaRa</td>
			<td>4030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, Absolute, ACS Grade</td>
			<td>Alfa-Aesar</td>
			<td>A9951</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Merck</td>
			<td>M-TMS-013-BKR</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Merck</td>
			<td>104008</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Bio-basic</td>
			<td>GB0235</td>
			<td></td>
		</tr>
		<tr>
			<td>GlycoBlue coprecipitant (15 mg/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Merck</td>
			<td>8.18766.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext rRNA Depletion Kit</td>
			<td>New England Biolabs</td>
			<td>E6318</td>
			<td>rRNA Depletion Kit</td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal rabbit IgG</td>
			<td>Cell signaling technology</td>
			<td>2729S</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR forward primer (RP1)</td>
			<td></td>
			<td></td>
			<td>5&#39;-AAT GAT ACG GCG ACC ACC GCG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA-3&#39;</td>
		</tr>
		<tr>
			<td>PCR index reverse primer (RPI)</td>
			<td></td>
			<td></td>
			<td>5&#39;-CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA-3&#39;</td>
		</tr>
		<tr>
			<td>PCR tubes with flat cap, 0.2 mL</td>
			<td>Axygen</td>
			<td>PCR-02-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate bufered saline (PBS) Tablet</td>
			<td>TaKaRa</td>
			<td>T9181</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion high-fidelity DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530</td>
			<td>High-fidelity polymerase</td>
		</tr>
		<tr>
			<td>PlateFuge microcentrifuge with swing-out rotor</td>
			<td>Benchmark</td>
			<td>c2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polynucleotide kinase (PNK)</td>
			<td>TaKaRa</td>
			<td>2021A</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail set III</td>
			<td>Merck</td>
			<td>535140-1MLCN</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant, PCR Grade</td>
			<td>Sigma-Aldrich</td>
			<td>3115879001</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR primer sequence: CO1 Heavy</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-GCCATAACCCAATACCAAACG-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: CO1 Light</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-TTGAGGTTGCGGTCTGTTAG-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: CO2 Heavy</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-CTAGTCCTGTATGCCCTTTTCC-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: CO2 Light</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-GTAAAGGATGCGTAGGGATGG-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: CO3 Heavy</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-CCTTTTACCACTCCAGCCTAG-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: CO3 Light</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-CTCCTGATGCGAGTAATACGG-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: CYTB Heavy</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-CAATTATACCCTAGCCAACCCC-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: CYTB Light</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-GGATAGTAATAGGGCAAGGACG -3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: GAPDH</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-CAACGACCACTTTGTCAAGC-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: ND1 Heavy</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-TCAAACTCAAACTACGCCCTG-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: ND1 Light</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-GTTGTGATAAGGGTGGAGAGG-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: ND4 Heavy</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-CTCACACTCATTCTCAACCCC-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: ND4 Light</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-TGTTTGTCGTAGGCAGATGG-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: ND5 Heavy</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-CTAGGCCTTCTTACGAGCC-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: ND5 Light</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-TAGGGAGAGCTGGGTTGTTT-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: ND6 Heavy</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-TCATACTCTTTCACCCACAGC-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>qPCR primer sequence: ND6 Light</td>
			<td></td>
			<td></td>
			<td>Forward/Reverse: 5&#8242;-TGCTGTGGGTGAAAGAGTATG-3&#8242;/5&#8242;-CGCAAATGGGCGGTAGGCGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>Random hexamer</td>
			<td>Thermo Fisher Scientific</td>
			<td>SO142</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Dnase I (Rnase-free) (5 U/&#956;L)</td>
			<td>TaKaRa</td>
			<td>2270A</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Rnase inhibitor (40 U/&#956;L)</td>
			<td>TaKaRa</td>
			<td>2313A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribo-Zero rRNA Removal Kit</td>
			<td>Illumina</td>
			<td>MRZH116</td>
			<td>rRNA Removal Kit</td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>FINEPCR, ROTATOR AG</td>
			<td>D1.5-32</td>
			<td></td>
		</tr>
		<tr>
			<td>RT primer sequence: CO1 Heavy</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGTTGAGGTTGCGGTCTGTTAG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: CO1 Light</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGGCCATAACCCAATACCAAACG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: CO2 Heavy</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGGTAAAGGATGCGTAGGGATGG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: CO2 Light</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGCTAGTCCTGTATGCCCTTTTCC-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: CO3 Heavy</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGCTCCTGATGCGAGTAATACGG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: CO3 Light</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGCCTTTTACCACTCCAGCCTAG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: CYTB Heavy</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGGGATAGTAATAGGGCAAGGACG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: CYTB Light</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGCAATTATACCCTAGCCAACCCC-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: GAPDH</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGTGAGCGATGTGGCTCGGCT-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: ND1 Heavy</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGGTTGTGATAAGGGTGGAGAGG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: ND1 Light</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGTCAAACTCAAACTACGCCCTG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: ND4 Heavy</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGTGTTTGTCGTAGGCAGATGG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: ND4 Light</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGCCTCACACTCATTCTCAACCC-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: ND5 Heavy</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGTTTGGGTTGAGGTGATGATG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: ND5 Light</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGCATTGTCGCATCCACCTTTA-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: ND6 Heavy</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGGGTTGAGGTCTTGGTGAGTG-3&#8242;</td>
		</tr>
		<tr>
			<td>RT primer sequence: ND6 Light</td>
			<td></td>
			<td></td>
			<td>5&#8242;-CGCAAATGGGCGGTAGGCGTGCCCATAATCATACAAAGCCCC-3&#8242;</td>
		</tr>
		<tr>
			<td>Siliconized polypropylene 1.5 mL G-tube</td>
			<td>Bio Plas</td>
			<td>4167SLS50</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dedecyl sulfate</td>
			<td>Biosesang</td>
			<td>S1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERase In Rnase inhibitor</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM2694</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III reverse transcriptase</td>
			<td>Thermo Fisher Scientific</td>
			<td>18080093</td>
			<td>Reverse transcriptase for library preparation</td>
		</tr>
		<tr>
			<td>SuperScript IV reverse transcriptase</td>
			<td>Thermo Fisher Scientific</td>
			<td>18090010</td>
			<td>Reverse transcriptase for qRT-PCR</td>
		</tr>
		<tr>
			<td>SYBR gold nucleic acid gl stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA ligase 1 (ssRNA Ligase)</td>
			<td>New England Biolabs</td>
			<td>M0204</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA ligase 2, truncated KQ</td>
			<td>New England Biolabs</td>
			<td>M0373</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf ThermoMixer C with ThermoTop</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thymidine</td>
			<td>Sigma-Aldrich</td>
			<td>T9250</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-borate-EDTA buffer (TBE)</td>
			<td>TaKara</td>
			<td>T9122</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultralink Protein A sepharose beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>22810</td>
			<td>Protein A beads</td>
		</tr>
		<tr>
			<td>Ultrasonicator</td>
			<td>Bioruptor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Bio-basic</td>
			<td>UB0148</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>DAIHAN Scientific</td>
			<td>VM-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene cyanol</td>
			<td>Sigma-Aldrich</td>
			<td>X4126</td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-32P-ATP (10 &#956;Ci/&#956;L, 3.3 &#956;M)</td>
			<td>PerkinElmer</td>
			<td>BLU502A100UC</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying rna interactors of protein kinase rna-activated during the mammalian cell cycle

蛋白激酶 rna 激活 (pkr) 是先天免疫反应蛋白的成员, 并能识别病毒 rna 的双链二级结构。当与病毒双链 rna (dsrna) 结合时, pkr 经历二聚化和随后的自磷酸化。磷酸化 pPKR (ppkr) 变得活跃, 并诱导真核起始因子 2 (eif2α) 的α亚基磷酸化, 以抑制全局平移。越来越多的证据表明, pkr 可以在生理条件下激活, 例如在细胞周期中或在各种压力条件下, 没有感染。然而, 由于缺乏捕获和分析 pkr 相互作用 dsrna 的标准化实验方法, 我们对 pkr rna 激活剂的理解有限。在这里, 我们提出了一个实验方案, 以专门丰富和分析 pkr 绑定 rna 在细胞周期中使用 hela 细胞。利用甲醛的有效交联活性固定 pkr-rna 复合物, 并通过免疫沉淀分离。然后可以进一步处理 pkr 联合免疫沉淀 rna, 以生成高通量测序库。pkr 相互作用的细胞 dsrna 的一个主要类别是线粒体 rna (mtrna), 它可以通过重链和光链 rna 之间的互补相互作用作为分子间 dsrna 存在。为了研究这些双工 mtrna 的散变性, 我们还提出了一种带特异性 qrt-pcr 的方案。我们的协议是针对 pkr 结合 rna 的分析而优化的, 但它可以很容易地进行修改, 以研究其他 dsrna 结合蛋白的细胞 dsrna 或 rna 相互体。

图 1显示了在细胞周期的 s 或 m 阶段逮捕 hela 细胞的过程示意图。对于 m 相抑制样本, 我们可以在显微镜下清楚地显示圆形细胞 (图 2a)。为了检查细胞周期抑制的效率, 可以使用流式细胞仪分析细胞的核含量 (图 2b)。图 3显示了免疫沉淀效率测试的代表性数据, d7f7 抗体显示出优于免疫沉淀 pkr 的能力。免疫沉淀效率的这种差...

Watch this Scientific Journal Video about Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle at JoVE.com

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

我们提出了利用 hela 细胞研究哺乳动物细胞周期中双链 rna 结合蛋白激酶 rna 激活 (pkr) 的 rna 相互作用的实验方法。该方法利用甲醛对交联 rna-pkr 复合物和免疫沉淀进行富集 pkr 约束 rna。这些 rna 可以通过高通量测序或 qrt-pcr 进一步分析。

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

该协议提供了一种方法，以全基因组方式识别RNA结合蛋白PKR的RNA相互作用者。它帮助我们更好地了解RNA结合蛋白对基因表达的转录后调控。该技术的主要优点是利用甲醛的高效交联来修复与RNA结合蛋白结合的RNase，并通过免疫沉淀来丰富它们。在神经退行性疾病（如帕金森氏症和阿尔茨海默病）患者中观察到激活PKR。识别PKR相互作用双链RNase将有助于我们更好地了解这些退行性疾病的发病机制。此方法可用于研究任何RNA结合蛋白的RNA相互作用器。特别是，我们认为，这种方法是有用的研究蛋白质，识别结构化的RNase，如双链RNase。在收获细胞之前，检查循环收集样本的均匀性。此外，使用最好的抗体优化免疫沉淀效率至关重要。首先，种子75万或100万个希拉细胞分别用于S-或M相逮捕样本。在37摄氏度和5%的二氧化碳下生长细胞24小时。用两毫摩尔胸腺素治疗细胞，并在37摄氏度下孵育18小时。用PBS清洗细胞两次。然后，加入新的介质，在37摄氏度下孵育9小时。对于S相逮捕细胞，用两毫摩尔胸腺素治疗细胞。对于M相逮捕细胞，治疗每毫升诺科达佐尔100毫微克的细胞。在37摄氏度下孵育15小时。对于 S 相样本，使用细胞刮刀收集细胞，然后转移到 15 毫升锥形管中。对于 M 相样本，点按细胞培养盘的侧面分离 M 相带细胞，然后将它们转移到 15 毫升锥形管中。接下来，在室温下以380g离心3分钟。取出上清液，用一毫升冷PBS重新暂停细胞。将细胞转移到1.5毫升微离心管中。在4摄氏度下将10，000克的细胞离心30秒，并完全去除超钠。要进行甲醛交联，首先在细胞中加入750微升0.1%的甲状甲醛，并在室温下孵育10分钟以固定细胞。然后，加入250微升的一摩尔甘氨酸，并在室温下孵育10分钟，以抑制反应。在4摄氏度下将10，000克的细胞离心30秒。完全去除超自然。接下来，重新使用一毫升的PBS。在4摄氏度下将10，000克的细胞离心30秒，然后取出上一代。重新使用400微升的fCLIP解液缓冲液，辅以0.2体积百分比蛋白酶抑制剂和2个体积百分比RNase抑制剂。在冰上孵育10分钟，并使用超声波器进行声波化。接下来，加入11微升的五摩尔氯化钠，将氯化钠浓度调整为150毫摩尔，并短暂旋转。然后，在4摄氏度下在21，130g下离心15分钟。将超自然剂转移到预冷却的 1.5 毫升微离心管中。为了进行免疫沉淀，首先将20微升蛋白质 A 珠加入 1.5 毫升微离心管。然后，用400微升的fCLIP解液缓冲液清洗珠子。在4摄氏度下将珠子在1000克下离心一分钟。接下来，去除上流液，小心地加入400微升的fCLIP解液缓冲液，然后通过反转三到四次轻轻重新填充珠子。重复洗涤步骤三次。最后洗涤后，完全取出超自然剂。将珠子重新填充在 300 微升的 fCLIP 解液缓冲液中。加入8.3微升的五摩尔氯化钠，将氯化钠浓度调整为150毫摩尔。加入10微升蛋白激酶RNA活性抗体，在4摄氏度的旋转器上孵育3小时。接下来，在4摄氏度下将珠子在1000克下离心一分钟。取出上流水线，加入400微升的fCLIP洗涤缓冲液，轻轻倒掉三到四次珠子。重复洗涤步骤两次。最后洗涤后，完全取出超自然剂。接下来，加入300微升的赖酸盐，并在4摄氏度的旋转器上孵育3小时。然后，在摄氏4度的1000克下将珠子离心一分钟。取出上流水线，加入400微升的fCLIP洗涤缓冲液，轻轻倒掉三到四次珠子。重复洗涤步骤三次。最后洗涤后，完全取出上经液。接下来，添加 300 微升的 fCLIP 洗脱缓冲液。在25摄氏度的热混合器中孵育3小时，从珠子中培育蛋白激酶RNA。在室温下在1000克下离心，将超自然剂转移到清洁的硅化管中。最后，加入300微升的蛋白酶K，在65摄氏度下孵育过夜。使用 FACS 检查了S或M相的 HeLa 细胞的细胞周期逮捕效率。D7F7抗体表现出对免疫沉淀蛋白激酶RNA激活的优越能力。西方对PKR免疫沉淀或总 HeLa lysate 的印迹分析仅显示一个与 PKR 大小对应的强带，表明 D7F7 抗体在识别 PKR 时具有高度特异性。使用不同的PKR抗体具有不同的免疫沉淀效率，导致不同的分类分布的地图测序读取。成功去除RRNA后，观察到PKR共免疫沉淀RNase的放射性同位素信号减少。PKR共免疫沉淀的RNA样本，与阴性对等对等相比，表现出线粒体RNase的强富集。斯特兰德特异性逆转录用于区分重链和轻链线粒体RNase，这些线粒体与PKR共同免疫，用于S或M相逮捕细胞。准备细胞周期逮捕样本最关键的一步是，您必须用 PBS 清洗细胞两次，并且必须只收集细胞柔和的均性或会受损的样本。对于道德流程，最关键的步骤是甲醛交联和数量。此过程之后，可以使用北印迹、qRT PCR 或高通量排序分析洗代的 RNA。这些实验可以识别绑定到 PKR 的 RNase 的类型和相对量。该技术可用于研究和识别RNA结合蛋白的RNA相互作用器，这将增进我们对RNA结合蛋白的转录后调控的理解。甲醛是危险的，因此需要谨慎处理固定、溶液和初始洗涤缓冲液。

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Assaying Protein Kinase Activity with Radiolabeled ATP

用放射性标记的ATP测定蛋白激酶活性

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

生长缓慢的社会细菌Myxococcus xanthus的全营养细胞周期的荧光活细胞成像

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

Studying Protein Import into Chloroplasts Using Protoplasts

原生质体在叶绿体中导入蛋白质的研究

The chloroplast is an essential organelle that is responsible for various cellular processes in plants, such as photosynthesis and the production of many ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

人类外周血神经细胞分离通过评估Rab10磷酸化来询问帕金森氏症相关的LRRK2激酶途径

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...